Protein Arginine Deiminase 4 (PAD4) is widely regarded in the Rheumatoid Arthritis (RA) community as the best new target for the development of a novel RA therapeutic. This enzyme hydrolyses Arg residues to form Citrulline and ammonia. The first goal of this proposal is to perform structure/activity relationships on F-Amidine, a fluoroacetamidine-based PAD4 inactivator discovered by the PI;and thereby identify a PAD4 inactivator with improved potency. The second goal is to take two approaches to identify a PAD4-selective inactivator. The first approach incorporates the haloacetamidine warhead into PAD-specific peptide substrates - substrate specificity studies will be used to identify these peptides. The second approach uses a focused library of haloacetamidine-containing compounds - inactivators with improved selectivity will be identified by screening the library with PAD4 and PAD2, a related enzyme that is the most widely expressed PAD isozyme. The third goal of this proposal is the use these haloacetamidine based compounds as the basis for synthesizing Activity Based Protein Profiling (ABPPs) Reagents. These ABPPs will not only be useful for characterizing the in vivo selectivity of PAD4 inactivators, but will also enable the isolation and/or enrichment of endogenous PAD4 from mammalian cell lines;and thereby facilitate the identification of the post-translational modifications that occur to this protein when it is activated in vivo. Once complete the proposed studies will lead to the identification of a potent and selective PAD4 inactivator that will represent a lead compound for the treatment of RA. In addition, the proposed compounds will be important probes that will be used to decipher the incompletely defined role of PAD4 in human cell signaling (e.g. gene regulation) and how (or if) dysregulation of these pathways contributes to RA. Lay Statement: The goal of this project is to develop inhibitors targeting Protein Arginine Deiminase 4. This goal is being pursued because the activity of this enzyme, when dysregulated, is believed to contribute to the onset and progression of RA, a disease that affects ~1% of the American population and causes a mean reduction in life expectancy of 5 to 10 years. The inhibitors described in this proposal not only represent lead compounds for the treatment of RA but will also be useful for further defining how this enzyme contributes to disease onset.